Repetitive hit-and-run immunoassay and stable support-analyte conjugates; applied to T-2 toxin

ABSTRACT

A repetitive immunoassay analytical method for determination of a free analyte is carried out by loading an affinity column of covalently bound analyte with tagged antibody, passing a continuous aqueous stream of carrier liquid over the column, introducing an aliquot of a sample to be analyzed for free analyte into the carrier stream upstream of the column, and monitoring the eluting carrier stream for a signal spike resulting from the presence of tagged antibody material released from the column by the application of free analyte in the analytical sample. Many samples may be analyzed by this method before the antibody-loaded affinity column needs to be regenerated. It is also disclosed that substrate-analyte conjugates of superior stability are produced by linking a substrate to a hydroxyalkyl analyte via an amino, hydrazide, or sulfide linking group replacing a hydroxy group. Such stable substrate-analyte conjugates are useful in the production and purification of antibodies, as well as in the repetitive immunoassay of the invention.

GOVERNMENT SUPPORT

The government has certain rights in this invention pursuant to contractN00014-84-C-0254, awarded by CRDC and administered by the Office ofNaval Research.

FIELD OF THE INVENTION

This invention relates to an immunoassay analytical procedure fordetermination of an analyte, and to substrate-analyte conjugates, andmore particularly to a repetitive hit-and-run immunometric assay and tosubstrate-analyte conjugates having particularly stable chemical linksbetween the substrate and analyte portions of the conjugates.

BACKGROUND OF THE INVENTION Introduction

The trichothecenes are a group of fungal by-products with a tetracyclic,sesquiterpenoid ring system. This group includes a number of highlytoxic compounds, one of which is T-2 toxin, a mycotoxin ##STR1##produced by fungi of the Fusarium species and isolated from mold foundon wheat, barley, oats, and corn (References 1-5). T-2 acts at thecellular level by inhibition of the initiation of protein synthesis(Reference 2). It exhibits some organ specificity, attacking mainly thehematopoietic organs, especially the bone marrow, resulting in markedleukopenia and cellular destruction (Reference 4). Adverse biologicalreactions are seen in both plants and animals. Phytotoxic effectsinclude stem and leaf scorching and stunting as well as wilting.Similarly, topical application of T-2 toxin to animal or human skincauses local irritation, inflammation, necrosis and desquamation of theepidermis (References 1, 2, and 5). Systemic administration of the toxinto laboratory mice causes a series of fluctuations in respiration andheart rate followed by a slow decline of both until death (References 1and 5). Epidemiological studies have shown that outbreaks of disease infarm animals in Britain and occurrences of "moldy corn toxicosis" incattle and commercial flocks of chickens were a result of exposure toT-2 toxin (References 2 and 3). Further, human intoxication, as a resultof moldy food consumption, has been traced to this source as well(Reference 9). Thus, because of the poisonous nature of T-2 toxin, andthe fact that the grains from which it has been isolated are used asboth food and feed, the demonstrated potential for exposure of bothhumans and animals is apparent. Consequently, sensitive and accuratemethods for analysis of T-2 toxin in food and feed are essential.

T-2 Assays

T-2 toxin has been measured by a varity of methods, including: (1)bioassay, (2) thin layer chromatography (TLC), (3) gas liquidchromatography (GC) with flame ionization (FID), electron capture (ECD)or mass spectroscopic (MS) detection, (4) high performance liquidchromatography (HPLC), and (5) immunoassay (References 6-15).

Some of the biological test systems for analysis of T-2 toxin are dermaltoxicity, inhibition of protein synthesis, and cytotoxicity. A list ofsome of the tests used and the detection limit of each for T-2 is givenin Table 1. The bioassay is highly sensitive, but it lacks specificity.As a result, one must assume that the biological response caused byadministration of a sample extract is due to T-2 toxin and not someother source (References 6 and 9).

                  TABLE 1                                                         ______________________________________                                        Biological Assays for T-2 Toxin                                               Biological System     Detection Limit                                         ______________________________________                                        Rabbit, dermal        0.01     μg/test                                     Pea seedling          <1       μg/ml                                       Brine shrimp          0.1-0.2  μg/ml                                       Mouse, intrapertioneal administration                                                               3.0-5.2  μg/kg                                       Human karyoblast      <1       μg/ml                                       Rabbit reticulocytes  0.03     μg/ml                                       ______________________________________                                         Reference 6, pg. 907                                                     

TLC has been a frequently used method for the identification andquantitation of trichothecenes (References 6 and 11). One of the majordifficulties associated with this method is low sensitivity. Thedetection limit for T-2 toxin is generally in the range of 0.1-0.2μg/spot (Reference 6). By comparison, bioassays for cytotoxicity candetect T-2 toxin at levels of 0.01-10 ng. It has therefore beensuggested that TLC be used in conjunction with bioassay to give a moresensitive and specific testing procedure (Reference 11). A shortcomingof that approach, however, is that more time and expense would beinvolved. An additional disadvantage of TLC is that closely relatedstructural analogues co-migrate and thus cannot be distinguished fromone another.

Gas liquid chromatography has been used to quantitate T-2 toxin inplasma as well as in foodstuffs (References 7-9, and 12). Both packedand capillary columns have been used in combination with FID, ECD, or MSdetection. Detection limits achieved by ECD are 2 to 5 fold lower thanthose obtained by FID (Reference 7). Mass spectroscopic detection isable to reach detection limits for T-2 toxin similar to those reached byECD (Reference 9). One of the most sensitive GC methods that has beendescribed combines ECD with a packed column in obtaining a detectionlimit of 25 ng/ml (Reference 12). Gas chromatographic analysis of T-2 isvery sensitive, and may have the advantage of allowing the simultaneousdetermination of several substances. However, performance of an assayfor T-2 by this means requires tedious sample preparation and the use ofexpensive, complex instrumentation. As with TLC, structural analogues aswell as matrix substances may be hard to separate from the analyte ofinterest. Finally, only one sample can be analyzed at a time by gaschromatography. Therefore, screening of large numbers of samples is timeconsuming and expensive.

HPLC has been used for the measurement of T-2 toxin and othertrichothecenes. However, detection of T-2 toxin by HPLC poses a problembecause T-2 lacks an ultraviolet or fluorescent chromophore. Thedetection limit for the analysis, when conducted using a differentialrefractometer detector, is in the microgram range and unsuitable evenfor grain analysis. Formation of the p-nitrobenzoate derivative of T-2toxin increases the sensitivity of the assay to 50 ng/kg, but requiresmore time and expense. Thus, as other assay systems show more promise,HPLC has seen limited use in quantitating T-2 toxin (References 7 and10).

Recently, immunoassays have been developed for the measurement of T-2toxin. These methods involve the simultaneous incubation of samples orstandards with a specific antibody against T-2 and a constant amount ofT-2 labeled with a signal group such as radioisotope or an enzyme. T-2in the sample and the labeled T-2 compete for binding to the specificantibody. The bound toxin is separated from the unbound (free) toxin byan appropriate technique, after which the amount of signal in the boundfraction is measured. The concentration of T-2 toxin in the sample isdetermined by reference to a standard curve (References 10 and 13-15).

In immunochemistry, hydroxy compounds sometimes require coupling to aprotein to provide immunogenic material, or to an affinitychromatographic surface to provide an affinity column for purificationof generated antibodies. Frequently this coupling must be performedunder aqueous conditions either because the protein will only toleratesuch conditions, or the hydroxy compound is water-soluble. When aqueousconstraints are present, succinic anhydride is used to activate thehydroxy compound (ROH), forming a hemisuccinate derivative (ROCOCH₂ CH₂CO₂ H) that now has a carboxyl functional group. The latter group thencan be activated under aqueous conditions for coupling onto an aminosite. A difficulty with this approach is that carboxylic acid esters aresusceptible to aqueous hydrolysis, and the hemisuccinate leash istherefore unstable. This problem has persisted for many years, limitingthe efficiency and performance of protein and affinity surfaces madefrom hemisuccinate compounds.

The antibody against T-2 has been produced previously by injectingrabbits with a protein T-2 conjugate such as BSA-T-2. In the conjugateemployed, T-2 was coupled to protein through a hemisuccinate ester link.This link is unsatisfactory, however, because it hydrolyzes in vivo,releasing T-2. Because of its low molecular weight, unconjugated T-2 isnot immunogenic and therefore does not stimulate antibody production inanimals. Moreover, unconjugated T-2 can cross cell membranes and entercells, where it can then exert its toxic effect, and in the case oflymphoid cells, inhibit the organism's immunological response (Reference15). Therefore, the stability of the BSA-T-2 conjugate employed forantibody production is of utmost importance. Recently, Hunter preparedmonoclonal antibodies in an effort to produce T-2 antibodies with hightiters (Reference 14). In this work, he showed that nonspecific orenzymatic hydrolysis of a hemisuccinate ester or amide linkage inBSA-T-2 conjugate released T-2, which then inhibited the immunologicalresponse. Thus, it is not surprising that polyclonal antibodies to T-2toxin produced in rabbits have suffered from the disadvantage of lowtiters, or that enormous effort and cost were required to obtain onlytwo positive hybridomas yielding an antibody of T-2. Although theantibodies produced by hybridomas were not of high affinity, furtherefforts to obtain better antibodies were discouraged until a more stableT-2 BSA conjugate became available.

Purification of antibodies against T-2 can be carried out by ammoniumsulfate precipitation and dialysis, affinity chromatography, or both. Inthe affinity chromatography approach, BSA-T-2 conjugate was attached toCNBr activated chromatographic material. The modified material waspacked into a small column, and the isolated immunoglobulin fraction ofascites fluid was passed slowly over the bed. Unbound immunoglobulinswere removed by extensive washing, then T-2 antibody was eluted.Unfortunately, because the BSA-T-2 conjugate employing a hemisuccinatelink is unstable, these columns rapidly lose their affinity for T-2antibody and are not cost-effective.

Both polyclonal and monoclonal antibodies have been used for theanalysis of T-2 toxin. Because of the nature of these assays, they arehighly specific for T-2, showing little or no interference from othertrichothecenes. While assays employing monoclonal antibodies have givenhigher detection limits (50 ng/tube) than those employing polyclonalantibodies (1 ng/tube) the unlimited production (>10 mg/ml in ascites)potential of monoclonal antibodies should make them valuable asanalytical reagents for T-2 analysis (References 13 and 15).

In summary, the advantages of immunoassays are: (1) they are sensitiveas well as specific, (2) they are adaptable to the performance of largenumbers of assays, (3) they are relatively easy to perform, and (4) theydon't require the use of expensive or sophisticated instrumentation. Onthe other hand, their sensitivity is limited by the poor quality ofantibody that has thus far been available, and the unavailability of astable T-2 affinity column for purifying this antibody.

Immunoassay as an analytical method

Immunoassays have been popular for measuring trace quantities ofsubstances (analytes) since Berson and Yalow described the method in1958. Most immunoassays use labeled analyte and depend upon the abilityof unlabeled analyte (An) to inhibit the binding of labeled analyte(*An) to specific antibody (Ab), when a limited amount of the antibodyis present (FIG. 1). After incubation, the reaction reaches orapproaches equilibrium. At that time free *An is separated from antibodybound *An and the amount of signal present in one or the other isdetermined. The analyte concentration in the sample is determined bycomparison of the decrease in *An binding produced by free An in thesample to that of a standard curve produced by adding known amounts ofAn to the assay system. In variations of this assay system labeledantibodies have been used instead of labeled antigen. Such assays arecalled immunometric assays.

Separation of free analyte from bound can be accomplished by a number ofprocedures. The most common method is the use of nonspecific adsorbents.Free analyte can be adsorbed from solution by a substance such ascharcoal. This method is simple, inexpensive, and fast. Nevertheless, ithas certain disadvantages. In some cases, bound as well as free labeledanalyte may be adsorbed, and in other cases the affinity of theadsorbent for the analyte may be so high that the equilibrium reactionis disturbed.

Another method of separating free analyte from bound is nonspecificprecipitation. Salt or organic solvents are added to the equilibriummixture to precipitate the antibody fraction. Disadvantages of thismethod include: (1) variation in results caused by improper mixing, (2)inaccuracies in quantitation caused by co-precipitation of analyte, and(3) high binding blanks caused by nonspecific adherence of labeledanalyte to precipitates.

A third method for separating free analyte from bound is the doubleantibody method. This method depends on the ability of a second antibodyto bind to soluble analyte-antibody complexes, causing precipitation ofthe entire complex. This second antibody is produced so that its bindingis directed toward antigenic sites on the first antibody outside of thesites that combine with analyte. Very sensitive measurements arepossible using this system and it is applicable to a large number ofassay systems. Despite this broad applicability some pitfalls of thismethod exist. They are: (1) the amount of precipitate is very small andis subject to solubilization during washing if the reaction is notmaintained at 4° C., (2) the concentration of first antibody must behigh enough to give a precipitate when the second antibody is added, and(3) the titer of the first antibody must be higher than about 1:50 orsuch a large amount of second antibody must be used that it would beprohibitive.

Free and bound analyte have also been separated on the basis of theirdifference in electrophoretic mobility and molecular size. These methodshave seen limited use because they require expensive equipment, majorexpenditures of time, and some exposure to hazardous conditions.

Finally, immunologically specific adsorbents have been prepared forseparations of free analyte from bound. Either analyte or antibody canbe immobilized on a solid surface through a covalent or noncovalentbond. This absorbent is added to the assay where it removes eitheranalyte or antibody from solution, thus facilitating the separation ofbound and free analyte. It is preferable to covalently bond the reactantto the surface to prevent it from leaking from the surface intosolution. Systems such as this have the advantage of being simple andrapid. However, using this system, one may not always be able to obtainreproducible results, establishment of equilibrium may take many hoursin some systems, assays may be less sensitive than comparable solutionphase assays, and antibody affinity may fall significantly upon bindingto a solid surface.

Traditionally, intact antibodies have been used for immunoassays.However, in the past few years, antibody fragments obtained by enzymedigestion (for example, F(ab')₂ and Fab) and in some instancessulfhydryl reduction (Fab') have been used. One such system used anFab'-β-galactosidase conjugate with an affinity column for theseparation of free analyte from bound (Reference 16). Digoxin was themodel analyte. The analysis was conducted as follows: samples orstandards of digoxin were mixed with an excess of Fab'-β-galactosidseconjugate and this mixture was incubated, after which it was passedthrough a column of immobilized digoxin analogue. Following sampleelution, the amount of Fab'-β-galactosidase in the eluent was measured.The amount of signal in the eluent is directly related to theconcentration of digoxin since excess monovalent Fab'-β-galactosidase isretained by the column. In this system it is to be noted that monovalentantibodies are superior to divalent ones, in that assay sensitivitywould be limited for divalent antibodies because a divalent antibodyhaving only one binding site filled would be retained by the affinitycolumn (Reference 16). This assay system is fast, sensitive andadaptable to automation (Reference 17). There are, however, somedisadvantages to this analysis scheme. First, the dissociation rate forthe analyte-antibody complex must be slow relative to the separationstep. If it is not, analyte will dissociate from the antibody complexand allow binding of the released antibody to the affinity columnresulting in a reduction in signal elution and a loss of sensitivity.Second, there is an inherent background signal that appears to bevariable from lot to lot of Fab'-β-galactosidase (References 16 and 17).This contributes a variable noise to the system, limiting its accuracy,precision and sensitivity. Third, an excess of Fab' conjugate must beused for each assay tube. At very low concentrations of analyte (1×10⁻¹¹mol/L) a 100 to 1000 fold excess of Fab'-β-galactosidase must be used toobtain a usable analysis (Reference 16). This would mean the expenditureof large quantities of conjugate. Fourth, the need to effect a thoroughaffinity separation may place a limit on the degree to which the columncan be miniaturized. Finally, the affinity separation column is notreused, making the assay more expensive to perform and limiting itsability to be miniaturized for in-the-field and in-the-office testing.

Affinity Chromatography

Affinity chromatography is a preparative technique for purifyingbiological molecules, and has seen little or no use for quantitativeanalysis in analytical biochemistry. It relies on the biospecificrecognition of two biomolecules that form a specific complex. One ofthese, selected as the ligand, is convalently immobilized on achromatographic surface. When a solution of the other is passed throughsuch an affinity column, the complementary molecule is selectivelyretained because of the biospecific complex it forms with theimmobilized ligand partner. After residual substances from the sampleare washed out of the column with buffer, the elution conditions arechanged so that the biospecific interaction is disrupted and the targetsubstance of interest is eluted from the column. Most commonly, this isachieved through a general change in the composition of the buffereluent such as a change in its pH, ionic strength, content of organicsolvent, or presence of a denaturant such as urea. Sometimes ligandelution is performed, in which the biospecific substance to be purifiedis eluted by the addition of the free ligand to the eluent.

The practice of affinity chromatography is a powerful but imperfecttechnique. Because of nonspecific binding of contaminants to the column,the column may have a short lifetime or may not fully purify the targetsubstance. The lifetime of the column may also be limited by aninstability of the attached ligand, either chemical breakdown orleakage. This problem is more severe when delicate biomolecules areimmobilized on the column. The use of general elution conditions such asthe change in pH may inactivate some of the target substance, as bydenaturation. Ligand elution may not be attractive since free ligand maybe expensive, or may not effectively elute a high affinity analyte. Alsoligand elution requires an additional step to remove it from the elutedtarget substance.

SUMMARY OF THE INVENTION

This invention relates to a repetitive immunometric assay method fordetermination of an analyte, the method including the steps of bondinganalyte covalently on a solid support to provide immobilized analyte;providing monoclonal antibody material corresponding to this analyte andcapable of noncovalent attachment to the bound analyte, the antibodymaterial also possessing a covalently bonded tag capable of providing ananalytically-useful signal; treating the immobilized analyte with thetagged monoclonal antibody material, to form an immobilized complex ofanalyte and tagged antibody material on the solid support; providing acontinuous stream of aqueous carrier liquid across the immobilizedcomplex; injecting into this stream of carrier liquid, upstream of theimmobilized complex, an aliquot of a solution to be analyzed for thefree analyte, which causes a complex of free analyte and tagged antibodyto be eluted from the immobilized complex in an amount related to theamount of free analyte present in the aliquot; monitoring the stream ofcarrier liquid, downstream of the immobilized complex, for analyticalsignal indicative of the presence and amount of the tag in the carrierstream; determining the amount of free analyte in the aliquot bycomparison of the signal produced with signals produced by addition ofknown amounts of free analyte to the system; and permissibly repeatingthe analysis using further aliquots of the same or different solutionsto be analyzed for the free analyte.

The invention also relates to compositions containing chemicallyimmobilized T-2 toxin or DAS toxin having the generalized formulae:##STR2## in which the structural formulae represent T-2 toxin and DAStoxin respectively, each mycotoxin being absent a hydroxy substituent atthe point where the linker unit is attached: the linker is ##STR3##--CONHNH--, or --S--; the substrate is a macromolecule such as aprotein, or a solid support of the sort generally used inchromatography; and the leash is a molecular chain connecting thesubstrate and the linking unit.

The repetitive immunoassay method is broadly useful for the analysis ofanalytes at high sensitivities, and the compositions are useful in theproduction and purification of antibodies against T-2 toxin and DAStoxin, and are also useful in the repetitive immunoassay method which isthe primary subject of this invention.

DESCRIPTION OF THE DRAWING

The invention will be better understood from a consideration of thedetailed description taken in conjunction with the drawing in which:

FIG. 1 is a diagram of the principle of radioimmunoassay.

FIG. 2 is an illustration of the concept of hit-and-run immunoassay.

FIG. 3 is an outline of the steps of the hit-and-run immunometric assaymethod.

FIG. 4A is an elution pattern of Fab'-fluorescein from a T-2 affinitygel.

FIG. 4B is an elution pattern of retained peak from FIG. 4A when appliedto a T-2 affinity gel.

FIG. 5 is an elution chromatogram showing purification of T-2 antibodyon a T-2 affinity column.

FIG. 6 shows signal pulses of fluorescence resulting from application of0 to 50 ng of T-2 toxin in the repetitive immunoassay.

FIG. 7 is a standard curve for detection of T-2 in the repetitiveimmunoassay using Fab'-fluorescein.

FIG. 8 is a standard curve for detection of T-2 in the repetitiveimmunoassay using Fab'-RNase.

FIG. 9 demonstrates the amount of Fab'-fluorescein-biotin bound to anavidin column, in comparison to Fab'-fluorescein as standard.

FIG. 10 demonstrates the amount of Fab'-fluorescein-biotin bound to aT-2 affinity column, in comparison to Fab'-fluorescein as standard.

FIG. 11 is a schematic showing preparation of a T-2 toxin BSA-conjugate.

FIG. 12 shows resistance of the enzymatic activity of Fab'-RNase toplacental inhibitor.

DETAILED DESCRIPTION

Traditionally, affinity chromatography has been used as a preparativetool. In this invention its analytical capabilities have been exploitedin developing a "hit and run" chromatographic technique (FIG. 2). Ananalytical chromatography column with a ligand covalently immobilized onthe surface of a solid support is loaded with labeled monovalentantibody which noncovalently complexes with the ligand. Addition of freeligand (e.g. T-2) elutes a pulse of labeled material from this column,and this is quantitated by the signal provided by the label, e.g.,flourescence. This column can thus act as a rapid, repetitive device fordetecting ligands such as T-2. Unknown specimens can be donesequentially without the need to reload the column except after manyanalyses have been done.

Referring now to FIG. 3, there is shown an outline of a repetitiveimmunoassay method. The first step, 10, is carried out by bonding theanalyte to be determined covalently to a solid support to immobilize it.The analyte may be anything which can be bonded to the solid support andwhich forms antibodies which can be chemically tagged. Examples ofanalytes to which the method is applicable are toxins such as T-2 andDAS, drugs such as digoxin, drug metabolites such asN-acetylprocainamide, hormones such as insulin, T₄, DHT, E₂, andinfectious disease agents such as viruses. A wide variety of solidsupports may be employed, including agarose, cellulose, Sepharose,Trisacryl, polyacrylamide, silica, glass, Immobilon Membrane, andplastic materials such as nylon, polymethacrylate, and polystyrene.

The next step, 12, involves providing monoclonal antibody materialcorresponding to the analyte and having a chemical tag capable ofproviding an analytically-useful signal. The monoclonal antibodymaterial may be intact monoclonal antibody, or preferably, a fragment ofa monoclonal antibody such as Fab'. Monoclonal antibodies are preferredover polyclonal antibodies because they can be prepared in bulk, theycan be more specific for the free analyte being determined, and they arehomogeneous. A wide variety of chemical tags are applicable, includingenzymes such as RNase, β-galactosidase, glucose oxidase, and horseradishperoxidase, which are monitored by their enzymatic activity;fluorophores such as a phycobiliprotein, rhodamine, fluorescein, andfluorescein plus biotin, which are monitored by their fluorescence;lumiphores such as luminol, isoluminol, and acridinium esters which aremonitored by luminescence; radioisotopes, monitored by theirradioactivity; dyes, monitored by absorbance; and electrophoric releasetags, monitored by gas chromatography. Such release tags and methods ofusing them are defined in pending U.S. patent application Ser. Nos.344,394, 591,262, and 710,318, hereby incorporated by reference.Preferred tagged antibody materials are Fab'-fluorescein, Fab'-RNase,and Fab'-lumiphore. In the tagged antibody material, the tag isgenerally attached to the antibody or antibody fragment via an amino,carboxyl, sulfhydryl, imidazole, or phenolic group on the antibody. Thetagged antibody material may contain one or more of these chemicallabels. These linkages and their methods of synthesis are known.

The next step, 14, involves treating the immobilized analyte with thetagged monoclonal antibody material to form immobilized complex ofanalyte and tagged antibody material on the solid support. An excess oftagged antibody material is employed to saturate the immobilizedanalyte, and subsequently the immobilized complex is washed thoroughlyto remove any unbound tagged antibody material or unreacted startingmaterials.

In the next step, 16, a continuous stream of aqueous carrier liquid isprovided across the immobilized complex.

In the following step, 18, an aliquot of the sample to be analyzed forfree analyte is injected into the flowing aqueous carrier streamupstream of the immobilized complex. As the carrier stream conveys thesample across the immobilized complex, the free analyte in the solutionaliquot complex with tagged antibody material, and elutes from theimmobilized complex with the carrier stream. The system operates in thisway because while immobilized analyte is held on the solid support, andtagged antibody material is immobile while it is complexed withimmobilized analyte on the support, the tagged antibody is in fact notpermanently attached to the immobilized analyte, but is in anequilibrium with free tagged antibody material in the aqueous carrierliquid. When free analyte comes in contact with free tagged antibodymaterial, a non-immobilized complex of free analyte and tagged antibodymaterial forms and elutes.

The next step, 20, involves monitoring the stream of carrier liquid,downstream of the immobilized complex, for signal indicative of thepresence and amount of the tag portion of the tagged antibody material.The particulars of such monitoring are a function of the tag employed tolabel the antibody material.

In the next step, 22, the amount of free analyte in the sample aliquotis determined by comparing the signal generated by the released tag withsignals generated upon application of known amounts of free analyte tothe system.

Finally, as indicated in step 24, analyses may be repeated or newanalyses may be performed by injecting succeeding aliquots of the sameor different samples, monitoring the signals generated upon applicatinof these successive samples, and determining the amount of free analytein each sample aliquot, as before. As the system contains a large amountof immobilized complex relative to the amount of free analyte in a givenaliquot of sample, many samples may be analyzed before the reservoir oftagged antibody material is depleted to an extent sufficient to detractfrom the analysis. Upon exhaustion or depletion of the reservoir ofimmobilized complex, the system is readily regenerated by treating thesolid support-immobilized analyte with excess tagged antibody materialas in step 14.

An important aspect of this method is that by providing a complex oftagged antibody material on an immobilized analyte, and employing acontinuous stream of carrier liquid over this material, the analyticalproblems caused by damaged antibodies in prior art immunoassayprocedures are avoided. In such prior art procedures, a samplecontaining free analyte is treated with excess tagged antibody and thenpassed over a column of immobilized analyte to remove the uncomplexedtagged antibody by an affinity-type separation and allow free complex ofanalyte.tagged antibody to elute from the column, the free analyte beingdetermined by determining the amount of chemical tag in the eluate.

While this prior art procedure is sound in theory, in practice itsuffers from the fact that the tagged antibody always has some "damaged"antibody present in addition to the desired tagged antibody in which theantibody is normal. This damaged tagged antibody can be of two kinds: afirst kind which does not bind to the immobilized analyte in theaffinity column and therefore immediately elutes from the column, and asecond kind which absorbs nonspecifically to the affinity column. Thenet result is that other factors besides the amount of free analytedetermine the amount of signal tag eluting from the column, therebycausing the determination of free analyte in an applied sample to beless sensitive, accurate and precise.

In the present procedure these problems are avoided by treating theimmobilized analyte with excess tagged antibody material to form acomplex of immobilized analyte and tagged antibody on the solid support,and washing this complex by means of the stream of aqueous carrierliquid. In this system, any damaged antibody either washes off theimmobilized analyte or binds essentially irreversibly, in either casecausing no analytical problems. Although a small amount of taggedantibody material washes out of the system continually, this constitutesa low-level constant background signal which is not an analyticalproblem. In fact, it can provide a steady-state background signal tohelp maintain the calibration and status of the system. Thus, when ananalytical sample is injected into the flowing stream of aqueous carrierliquid, the signal spike which elutes from the system quantitates thefree analyte injected onto the immobilized analyte-tagged antibodycomplex.

In an alternative embodiment, the free analyte may also move bydiffusion or electrophoretic migration across the immobilized complex,causing the movement of the analytical signal, which determines theamount of free analyte. In this case the analysis is also permissablyrepeated.

The "hit-and-run" immunoassay procedure described above requires analytecovalently bound to solid support, as well as antibodies correspondingto this analyte. The production of such antibodies in turn requires asuitable immunogen and means for purifying the immunoglobulins isolatedfrom a host animal in which the antibodies were produced. When theanalyte is a relatively small molecule, it is frequently not immunogenicby itself, and therefore formation of a suitable immunogen requiresattachment of the analyte to a carrier molecule, most generally amacromolecule such as a protein. Various techniques for accomplishingthis are known. Purification of the antibody produced by action of theprotein-analyte conjugate in the host animal is generally carried out byaffinity chromatography on a column of analyte covalently immobilized ona suitable solid support. The solid support on which the analyte iscovalently bound for purposes of affinity chromatographic purificationof antibody may be the same or different from that employed in thehit-and-run immunoassay procedure. As indicated above, varioustechniques for immobilizing analytes on supports are known. An improvedlink for joining an analyte to a support is described below, but it notrequired for the above-described hit-and-run immunoassay method, thoughit may be advantageously employed in that method.

Whether analyte is bonded to a solid support fo use in the hit-and-runimmunoassay or for affinity chromatographic purification ofimmunoglobulins, or to a macromolecule such as a protein for use as animmunogenic agent (immunogen), it is desirable for the chemical linkagebetween the substrate and the analyte to be stable, so thesubstrate-analyte conjugate does not degrade readily. It is an aspect ofthis invention that an amino, hydrazide, or sulfide functionalityemployed as the link between a substrate and an immobilized analyte andreplacing a hydroxy group on a hydroxyalkyl analyte possesses therequisite stability.

Thus, compounds useful but not required in the practice of thisinvention are conjugates of the form substrate-leash-linker-analytewhere the analyte portion of the conjugate is derived from ahydroxyalkyl analyte by replacement of the hydroxy group of thehydroxyalkyl portion of the analyte with the linker portion of theconjugate. In these hydroxyalkyl analyte-derived compounds, thefollowing definitions apply:

a. The substrate is a solid support or macromolecule, in particular,agarose, cellulose, Sepharose, Trisacryl, polyacrylamide, silica, glass,Immobilon Membrane, a polymeric material such as nylon,polymethacrylate, polystyrene, or a protein such as BSA.

b. The leash is a molecular chain attached to the substrate and to thelinker, and is either part of the substrate originally or is attached tothe substrate by an appropriate chemical reaction. In the event thesubstrate is a protein, the leash can be the C-terminal end, theN-terminal end, or a side chain containing an appropriate terminalfunctional group such as an amine. Where the substrate is a solidsupport the leash can be derived from a difunctional molecule of from 2to 10 carbon atoms with the functional groups being such reactivefunctionalities as carboxylic acids, carboxylic acid halides,carbohydrazides, carboxylic acid active esters, anhydrides, aldehydes,amines, epoxides, hydrazines, thiols, maleimides, alkyl halides, acylazides, etc. Examples of some suitable leash precursers are adipic acid,diaminooctane, adipic acid dihydrazide, mercaptoethylamine, succinicanhydride, ethylene diamine, hexanediamine, and glutaraldehyde.

c. The linker is ##STR4## --CONHNH--, or --S-- which replaces a hydroxygroup on the hydroxyalkyl analyte.

d. The analyte may be any hydroxyalkyl material which can be covalentlyconnected to the linker, particular examples being T-2 toxin and DAStoxin. Other exemplary analytes are E₂ and DHT.

These hydroxyalkylanalyte-derived materials are preferably formed byreactions between a nucleophile such as an amine, hydrazide, orsulfhydryl group on the leash to the substrate, and a derivative of theanalyte bearing a good leaving group such as a sulfonate, resulting indisplacement of the leaving group and formation of a stable covalentbond between the substrate and the analyte. This is shown in thefollowing equation: ##STR5## where the substrate and leash are asdefined above, the nucleophile is --NH₂, --CONHNH₂, or --SH; and theanalyte bears a leaving group such as a sulfonate, on an alkyl portionof the molecule.

The sulfonate ester employed as the leaving group may be any of thoseknown to the art, including tosylate, brosylate, nosylate, mesylate,triflate, nonaflate, and tresylate. The tresyl esters are particularlyadvantageous analyte derivatives. Other leaving groups known to the artwill also serve.

The tresyl esters of T-2 toxin and the closely related toxin DAS, havingthe structures ##STR6## respectively, are the preferred intermediates inthe preparation of T-2 and DAS-containing conjugates.

Tresyl Coupling

Tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride) is available asan activating agent for coupling amino and sulfhydryl compounds tohydroxymethyl chromatographic supports, as has been reviewed (K. Nilssonand K. Mosbach, Meth. Enz, 104, (1984) p. 56-69). A tresyl sulfonateester is formed on the chromatographic solid support, giving anactivated support (support--CH₂ OSO₂ CH₂ CF₃), and this in turn reactswith the amino or sulfhydryl compound (RNH₂ or RSH) to achieve covalentimmobilization of the latter, giving support--CH₂ --NHR or support--CH₂--S--R. The advantages of this reaction are that the activated supportis stable when stored at low pH; the activated support will reactefficiently with an RNH₂ or RSH under aqueous conditions at pH 7.5; andthe final linkages obtained are stable. Other activating agents forhydroxymethyl surfaces have not combined these desirable properties assuccessfully. Both Pharmacia and Pierce have made tresyl-activatedaffinity supports available commercially. Polyethylene glycol, a hydroxycompound, also has been tresyl-activated for coupling to a protein (K.Nilsson and K. Mosbach, Ibid.).

In other reported work, Mosbach, et al (Mosbach, K. H.; Furulund;Nilsson, K. G. I., Method of Covalently Binding Biologically ActiveOrganic Substances to Polymeric Substances, U.S. Pat. No. 4,415,665,Nov. 15, 1983) have described the activation of a hydroxy-containingpolymeric carrier by tresyl chloride or an analogous sulfonylhalogenide. This activated surface can then be reacted with abiologically active organic substance containing a primary or secondaryamino group, thiol, or aromatic hydroxy group. However, hydrazidecoupling to a hydroxyalkyl group activated with a sulfonyl halogenidehas not been described.

Tresyl-hydrazide coupling

In the present invention the problem of stable coupling of T-2 to aprotein or affinity surface is overcome by using tresylchloride-activated T-2 and specified nucleophilic groups on thesubstrate. As stated above, ordinarily tresyl chloride is used toactivate an affinity surface for coupling to an amino or sulfhydrylcompound. This invention has significantly broadened its use byactivating instead the T-2 hydroxy compound for coupling to a surface,thus bridging the distant worlds of activating an affinity surface andpreparing a novel derivative of T-2 for aqueous coupling. Also, theintroduction of a new coupling technique for a tresyl-activatedcompound, reaction onto a hydrazide site, has the advantage that theresulting alkyl-hydrazide linkage is noncharged at neutral pH, whereasan alkyl-amino linking gives a positive charge. The latter charge isgenerally undesirable because it not only imposes an ion exchange siteon the affinity material, complicating the chromatographiccharacteristics of the affinity material, but this charge significantlychanges the chemical nature of the hydroxy compound. This may interfere,for example, with the recognition of the latter compound in the attached(surface-immobilized) forms of this compound. It is important for bothpurification of a T-2 antibody (an antibody that specifically recognizesT-2), and for the use of such an antibody in an immunoassay for T-2,that surface-attached and free forms of T-2 are both recognized by theantibody.

Other conjugates useful in the practice of the invention are taggedfragments of monoclonal antibodies, Fab'-tag, where the antibodyfragment Fab' is derived from pepsin catalyzed cleavage of an antibody,and the tag is as previously defined. Particular Fab'-tag conjugates ofinterest are those in which the Fab' is a fragment of T-2 antibody andthe tag is one or more molecules of fluorescein, RNase, β-galactosidase,glucose oxidase, horseradish peroxidase, luminol, isoluminol, anacridinium ester, a release tag, or a dye. Also of interest is theconjugate Fab'-tag in which the Fab' fragment is derived from DASantibody and the tag is one or more of those listed above.

The substrate-leash-linker-analyte conjugates of the invention employingas the linker an amino, hydrazide, or sulfide linkage are not restrictedto use in the hit-and-run immunoassay procedure, but are broadly usefulin analytical biochemistry wherever stable conjugates of an analytechemically bonded to a substrate are needed. In particular, theconjugates in which the substrate is a macromolecule such as a proteinare generally useful as immunogenic agents (immunogens) to induceformation of antibodies in an animal against the covalently boundanalyte. Although substrate-analyte conjugates are known for thispurpose and the procedures for antibody production and isolation arealso known, the improved conjugates produced by employing as the linkingunit an amino, hydrazide, or sulfide group in place of a hydroxy groupon a hydroxyalkyl analyte are worthwhile.

Similarly, the substrate-leash-linker-analyte conjugates of theinvention are also more broadly useful in the area of affinitychromatography for the purification of antibodies. Although affinitychromatography for this purpose is known, the linkages employed in thepresent conjugates are more stable than those of the prior art andtherefore produce more stable and long lasting affinity chromatographysupports for hydroxyalkyl substances.

Employment of an ##STR7## --CONHNH--, or --S-- linkage to join asubstrate and a hydroxyalkyl analyte is advantageous because such linksare stable relative to those of the prior art. Thus, protein-analyteconjugates so linked employed in antibody production do not hydrolizereadily in vivo, so produce antibodies efficiently. Similarly,chromatographic columns packed with covalently bound antibody soimmobilized do not lose bound analyte readily, and so retain theiraffinity properties.

EXPERIMENTAL SECTION Materials

Bovine pancreatic ribonuclease A (Type IIIA) (RNase), polycytidylic acid(Poly C), cytidine 2'-3' cyclic monophosphate, bovine serum albumin(BSA), N-ethylmaleimide, 2-mercaptoethylamine, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), trinitrobenzene sulfonic acid (TNBS), andiodoacetamide were obtained from Sigma Chemical Co. (St. Louis, Mo.).Citraconic anhydride and mercaptoethanol were from Eastman OrganicChemical (Rochester, NY). Dimethylformamide (DMF), dimethyl sulfoxide(DMSO), and immobilized pepsin were purchased from Pierce Chemical Co.(Rockford, Ill.). Fluorescein isothiocyanate was obtained from AldrichChemical Co. (Milwaukee, Wis.). N-γ-maleimidobutyryloxysuccinimide(GMBS) and Biotin-X-NHS were from Calbiochem-Behring (Layolla, Ca.).Lanthanum acetate was purchased from Alfa Products (Danvers, Ma.).Ascites fluid containing anti T-2 monoclonal antibody was provided byKen Hunter and was prepared by the procedure published in his paper(Reference 14). 3-[3H]T-2 toxin was from Amersham (Arlington Hts., Ill).T-2 Toxin was obtained from the Myco-Lab Company (Chesterfield, Mo.).Buffer salts were purchased in the highest purity available from FisherScientific (Medford, Ma.). Agarose adipic acid hydrazide was obtainedfrom P-L Biochemicals (Milwaukee, Wis.). 2,2,2-Trifluoroethanesulfonylchloride was purchased from Aldrich Chemical Company (Milwaukee, Wis.).

Preparation of Fab',Fab'-fluorescein, and Fab'-RNase, and Size exclusionFPLC chromatography

Fab' was prepared by digesting antibody (obtained and purified asdefined below) with pepsin followed by reduction withmercaptoethylamine. This product was reacted directly withmaleimido-citraconyl-RNase (see below), which is de-citraconylatedduring the conjugation to Fab' to give Fab'-RNase. For the preparationof Fab'-fluorescein, the reduced Fab' was capped with iodoacetamide andthen reacted with fluorescein isothiocyanate.

The purity and molecular weight of the antibody fragments andderivatives were determined on a Superose 12/30 column (Pharmacia) usinga Pharmacia FPLC. The elution was carried out in 0.1M NaH₂ PO₄, 6Mguanidine HCl pH 7.5 at 0.3 ml/min, 0.2 AUFS. The amount of proteininjected was between 50 and 100 μg. Unless otherwise stated, theconcentrations of antibody and its fragments, F(ab')₂ and Fab', werecalculated from the absorbance at 280 nm by using an extinctioncoefficient of 1.5 cm² /mg for the antibody and 1.48 cm² /mg for F(ab')₂and Fab'.

Size exclusion FPLC was used to monitor the enzyme cleavage, chemicalreduction, and conjugation reactions of T-2 monoclonal antibody. Themonoclonal antibody eluted as a single peak with a molecular weight of158,000. After incubation of the antibody with immobilized pepsin for 20hr, a shift in the peak maximum was observed to M.W. 103,000,corresponding to F(ab')₂. Treatment of F(ab')₂ with mercaptoethylaminefollowed by acetylation with iodoacetamide resulted in the disappearanceof the peak at MW 103,000 and appearance of a new peak at MW 57,000(Fab'). Finally, both Fab-RNase and Fab'-fluorescein were similarlycharacterized and found to have MW of 56,000 and 54,000 respectively.

Preparation of Fab'-fluorescein conjugate

Anti T-2 monoclonal antibody was purified from mouse ascites fluid byprotein A affinity chromatography using the Bio Rad MAPS procedure(Reference 21). After purification, the antibody was dialyzed overnightvs 4 l of 0.02M sodium acetate pH 7.0. The pH of the solution waslowered to 4.2 with glacial acetic acid and 0.25 ml of immobilizedpepsin were added for each 10 mg of antibody to be digested. Digestionwas stopped after 20 hr by removing the pepsin beads and raising the pHof the solution to 7.0 with 1N NaOH. The digest was reapplied to theprotein A column to remove residual undigested antibody and pFcfragments. The unretained fraction from the protein A chromatography,containing the F(ab')₂ fragment, was dialyzed overnight vs 4 l of 0.1MNaH₂ PO₄, 5 mM EDTA pH 6.5 and concentrated from 40 to 20 ml ofrepetitive use of a Centricon 30 Microconcentrator (Amicon). Reductionof the F(ab')₂ fragments was accomplished by addition of 0.1 ml of 0.1Mmercaptoethylamine for each milligram of F(ab')₂ and incubation at 37°C. for 90 minutes. The sulfhydryl groups on the resulting Fab' fragmentswere alkylated by adding 0.1 ml of 0.2M iodoacetamide per milligram ofprotein and mixing for 60 min at room temperature. The mixture wasdesalted on a Sephadex G-25 column (1.6×40 cm) equilibrated with 50 mMNaH₂ PO₄ 150 mM NaCl pH 7.2. The alkylated Fab' fragments which elutedin the void volume were pooled, the pH was adjusted to 9.5 with 1Nsodium hydroxide, and 6 mg of fluorescein per milligram of Fab' wereadded. After mixing for 2 hr at room temperature, the labeled proteinwas purified on a Sephadex G-25 column (1.6×40 cm). The fractionscontaining the labeled protein were pooled and concentrated to a volumeof 15 ml in a Centricon-30 Microconcentrator (Amicon).

Degree of fluorescein labeling of Fab'

The number of fluorescein molecules coupled to Fab', givingFab'-fluorescein, was calculated from the absorbances at 280 nm and 495nm as described by The and Feltkamp (Reference 22). Calculations wereperformed according to the following formula: flourescein/protein=(2.87A₄₉₅ /A₂₈₀ -0.35 A₄₉₅). From this it was determined that the fluoresceinconjugate Fab'-fluorescein contained 4.7 moles of fluorescein per moleof Fab'.

Preparation of Maleimido-citraconyl-RNase

In order to protect amino groups critical for enzymatic activity,ribonuclease was treated with a 30 fold molar excess of citraconicanhydride over the number of amino groups. One hundred twenty milligrams(8.8 μmol) of RNase were dissolved in 6 ml of 0.12M HEPES pH 7.5 and23.7 μl (264 μmol) of citraconic anhydride were added. This solution wasmixed for 10 min at room temperature while the pH was maintained at 7.0with 1N NaOH. This reaction blocks approximately 9 of the amino groupsof the native RNase. Subsequently, 100 μl of DMF containing 60 mg (215μmol) of GMBS were added and mixing was continued for 30 min to blockmost of the remaining amino groups with GMBS. Then, the reaction mixturewas desalted using a PD-10 column with 0.005M sodium acetate pH 6.0 asthe eluent. The void volume fractions containing the protein were pooledand lyophilized.

Number of amino groups modified by GMBS in maleimido-citraconyl-RNase

Twenty-five μl (0.35 μmol) of mercaptoethanol in 0.1M KH₂ PO₄ pH 7.0were added to 1.0 ml (0.22 μmol) of citraconylated GMBS RNase (1.6maleimide residues/RNase) in the same buffer. The reaction mixture wasstirred for 10 min at room temperature and subjected to gel filtrationon a PD-10 column with 0.12M HEPES pH 7.5 as eluent. Deblocking of aminogroups modified by citraconic anhydride was accomplished by adjustingthe pH of the product from the PD-10 column to 2.5 with 0.1M HCl andmixing at 37° C. for 2 hr. The number of modified amino groups, relativeto native RNase, was determined by means of the TNBS test (Reference 18)before and after deblocking. Briefly, 100 μl of protein solution (0.5mg/ml) were added to 900 μl of 0.1M NaBO₄ pH 9.3 followed by 100 μl of0.03M TNBS in water. Samples were incubated for 30 min at roomtemperature and the absorbance was measured at 340 nm. It was thusdetermined that about 10 of the amino groups were blocked by thecombination of citraconic and GMBS blocking groups. After HCl deblockingto remove anhydride blocking groups, 1.4 amino groups were found to bestill modified by GMBS. This result corresponds well with the number ofmaleimide groups that were measured by the Ellmans reaction, reportedbelow (Reference 19).

Determination of maleimide groups in citraconylated GMBS RNase

The number of maleimide groups incorporated per mole of RNase wasdetermined by a modification of the method of Ellman, et al, (Reference19). Mercaptoethanol standards (5-100 nmol) and RNase samples (10-70nmol) were prepared in 1 ml of 0.1M KH₂ PO₄ 20 mM EDTA pH 7.2. Onehundred nanomoles (1 ml) of a freshly prepared solution of themercaptoethanol in the same buffer was added to all tubes. Afterincubation at 37° C. for 1 hour, 3 ml of 0.4M TRIS HCl, 20 mM EDTA pH8.9 and 0.1 ml of 10 mM DTNB (in methanol) were added. The tubes wereincubated for 10 minutes at room temperature, after which the absorbanceat 412 nm was measured. Approximately 1.2 maleimide groups per RNasemolecule were found.

Preparation of FAB'-RNase conjugate

Anti T-2 monoclonal antibody was digested and reduced as described abovefor preparation of the fluorescein conjugate, with the exception thatthe sulfhydryl groups of the Fab' fragment were not alkylated withiodoacetamide. Fab'(7.4 mg) in 25 ml of 0.1M NaH₂ PO₄ 5 mM EDTA pH 6.5was mixed with citraconylated GMBS RNase (45.64 mg). This mixture wasstirred overnight at 4° C., concentrated to a volume of 10 ml byultrafiltration on a Centricon-30 Microconcentrator, and purified by gelfiltration on a column (1.6×40 cm) of Sephadex G-75 with 50 mM TRIS HCl150 mM NaCl pH 7.2 as the elution buffer. The fractions containing theFab'-RNase conjugate were pooled and the volume was reduced to 10 mlusing Centricon-30 membrane filters. Fab'-RNase was found to beresistant to inhibition by placental inhibitor, as shown in FIG. 12.

Determination of RNase activity in maleimido-citraconyl RNase andFab'-RNase conjugate

Monomeric substrate: One milligram of cytidine 2',3' cyclic phosphatewas dissolved in 10 ml of 0.5M TRIS HCl, 5 mM EDTA pH 7.5. Eight hundredmicroliters of this solution were pipetted into 1 ml cuvettes, 100 μlsamples of RNase (10-100 μg) were added, and the change in absorbance at284 nm with time was measured.

Polymeric substrate: RNase standard (30-1000 pg) and sample solutionswere prepared in 0.5M TRIS HCl, 5 mM EDTA, 0.1% BSA pH 7.5. Fiftymicroliters of each solution was added to 100 μl of 0.5% poly C(w/v inwater). The tubes were incubated at 37° C. for 30 min, placed in an icebath, and 50 μl of ice cold 14 mM lanthanum acetate in 24% perchloricacid were added. After a 15 min incubation the tubes were centrifuged at1700×g for 20 min at 4° C. A 100 μl aliquot was withdrawn from thesupernatant, diluted 10 fold with water and the absorbance measured at260 nm.

This will be useful for distinguishing the enzymatic activity ofFab'-RNase from background RNase activity that may be encountered in asample undergoing chemical analysis.

The enzymatic activity of maleimido-citraconyl RNase towards 2'3'cytidine cyclic phosphate after deblocking increased from 51% to 94%.Moreover, up to 68% of the acitivity towards poly C was recovered. Theenzymatic acitivity was measured after reaction of the maleimide RNasederivative with mercaptoethanol to avoid cross-linking during theactivity study.

The RNase activity of the Fab'-RNase conjugate toward both monomeric andpolymeric substrates was measured. The amount of activity on a molarbasis of this conjugate, relative to that of native ribonuclease, is 5%with 2'3' cytidine cyclic monophosphate as a substrate and 2% withpolycyticylic acid substrate.

Preparation of 14 C succinylated ribonuclease

Ribonuclease (200 mg; 1.617 μmol) was dissolved in 35 ml of DMSOcontaining N-methylmorpholine (1634 μmol) and dimethylaminopyridine(1.47 μmol). Succinic anhydride (1.25 mg; 12.55 μmol) was dissolved in 1ml of dry DMSO and added to 250 μCi (2.15 μmol) of ¹⁴ C succinicanhydride. The succinic anhydride solution was vortexed and added to theribonuclease mixture. The ¹⁴ C succinic anhydride vial was washed oncewith DMSO and this wash was added to the reaction mixture. After shakingovernight at room temperature the reaction mixture was dialyzed for 48hr vs 5×4 l distilled water. The dialysate was lyophilized and stored at-20° C.

Number of RNase molecules per mole of Fab'

Citraconylated GMBS RNase was prepared according to the procedurepreviously described, except a mixture of ¹⁴ C succinylated and nativeRNase with a specific activity of 164 cpm/μg was used. ¹⁴ C succinylatedcitraconylated GMBS RNase (2.7 mg) was dissolved in 0.1M NaH₂ PO₄, 5 mMEDTA pH 6.5 containing 5.4 mg Fab'. The reaction was allowed to proceedovernight on a Nutator at 4° C. The conjugate was isolated by gelfiltration on a column (1.6×80 cm) of ACA 44 (LKB) with 10 mM TRIS HCl,150 mM NaCl pH 7.2 as the eluent, and showed a specific activity of 63cpm/μg. The number of moles of RNase per mole of Fab' was calculatedfrom the concentration of protein per ml of conjugate as determined bythe Pierce assay (Reference 23) and the number of counts of ¹⁴ C asdetermined by liquid scintillation. By this method it was determinedthat 1.1 moles of RNase are coupled to 1 mole of Fab'.

Binding of Fab'-fluorescein and Fab'-RNase to T-2 affinity gel

Two hundred microliters of T-2 affinity gel (see below) were packed intoa 1 ml Supelclean (Supelco) polypropylene filtration column having apolyethylene frit. The column (0.5×1 cm) was equilibrated with 10 ml of0.01M TRIS HCl pH 7.5 after which 2 ml (0.8 A₂₈₀ /ml) of eitherFab'-fluorescein or Fab'-RNase were applied. The column was washed with10 ml of equilibration buffer and eluted with 10 ml of 1N NH₄ OH pH11.6. Absorbance was monitored at 280 nm and fractions were collected inacetic acid to maintain the pH at 7.0. Samples were taken to measureprotein recovery and the rest of the pooled fractions were recycledthrough the T-2 affinity column.

A portion of each conjugate (23.4% of Fab'-fluorescein, 40% ofFab'-RNase) was not retained by the column, indicating some antibodybinding sites for T-2 had been damaged or blocked during the digestionor the chemical modification process. Of the total Fab'-fluoresceinapplied to the column, 15% was eluted with 1M NH₄ OH. This left 62% ofFab'-fluorescein unaccounted for. Corresponding work with Fab'-RNaseresulted in 39% being eluted by 1M NH₄ OH and 21% unaccounted for.

Subsequently, the eluted fractions of both the Fab'-fluorescein and theFab'-RNase were reapplied to the column and again eluted with 1M NH₄ OH.As before, a portion of each conjugate was not retained. This furtherindicated that some damage occurs to the T-2 binding site of bothconjugates under the elution conditions used. This is shown in FIG. 4for Fab'-fluorescein. Fab'-RNase gave similar results. Similar resultswere obtained where the Fab'-fluorescein was eluted from the affinitygel with excess T-2, dialyzed exhaustively to remove T-2, and re-appliedto the T-2 affinity column.

Preparation of [³ H]T-2/T-2 mixture

Five hundred microliters of [³ H]T-2 (11 Ci/mmol) were mixed with 45 mgof T-2 after which the solvent was evaporated and the resulting powderwas dried using high vacuum. Subsequently the mixture was recrystallizedfrom benzene/hexane. The yield was 34 mg (76% recovery). The specificactivity of the crystallized material was 27 cpm/μg.

Preparation of T-2 Toxin tresyl ester

To a solution of 34 mg (0.072 mmol) 3-[³ H]T-2 toxin (25,000 dpm/mmol)in 2 ml of dry actone, 35.5 ml (0.437 mmol) pyridine and 48.5 ml (0.439mmol) 2,2,2-trifluoroethanesulfonyl chloride were added at 0° C. Thereaction mixture was stirred for 3 hr. under nitrogen at roomtemperature. At this time the reaction was complete as determined by TLC(toluene:ethyl acetate:formic acid, 6:3:1 v/v/v). The solvent wasevaporated to dryness and the residue was redissolved in 15 ml of ethylacetate. The organic layer was washed three times with cold 5 mM HCl,once with cold water, and dried over anhydrous sodium sulfate. Thesolvent was evaporated and the product was dried under high vacuum,giving a white powdery material that was stored at -15° C.

Preparation of T-2-toxin-adipic acid hydrazide-agarose affinity gel

Agarose-adipic acid hydrazide gel (5 ml, PL Biochemicals) containing 1-6μmole of hydrazide/ml of gel was washed with 20 ml each of water and amixture of DMSO/0.01M potassium phosphate buffer pH 7.2 (70/30 (v/v)).The gel was suspended in the same mixture containing 18.4 mg (30 μmol)of [³ H]T-2 toxin tresyl ester. After shaking for 3.5 hr at 4° C., thegel was washed on a sintered glass funnel with 20 ml of the aboveDMSO/buffer, 20 ml of methanol/buffer (70/30 v/v), and finally with 20ml of buffer. The gel was stored in the DMSO/buffer mixture at 4° C. Theamount of T-2 toxin bound to the agarose gel was ≧0.2 μmol/ml of gelbased on the content of radioactivity.

the T-2 toxin-agarose was found to bind 6 mg of anti T-2 antibody per mlof gel, which antibody could be fully eluted with T-2. The antibodycomplexed on the T-2 column did not elute with a variety of chaotropicagents. However, non-specific elution was achieved with 1M ammoniumhydroxide, pH 11.6, at 4° C. After dialysis, 70-80% of the total proteinapplied to the column was recovered as fully active antibody. A typicalelution profile is shown in FIG. 5.

Stability of T-2 agarose affinity gel

The stability of T-2 toxin agarose gel to 1M ammonium hydroxide pH 11.6was investigated according to the following procedure. Excess antibodywas applied to the same T-2 affinity gel column (0.5×1.3 cm) in threeconsecutive runs, each time eluting with 1M ammonium hydroxide. Aftereach run, the eluent was dialyzed and the amount of antibody eluted wasmeasured by both the Lowry protein assay (Reference 20) and absorbanceat 280 nm. No decrease in the binding capacity of the T-2 affinitycolumn was observed (0.94±0.08 mg).

Purification of anti T-2 from ascites by T-2 affinity chromatography

The following experiment was performed in a cold room at 4° C. T-2affinity gel (900 μl) was packed into a 1 ml Supelclean (Supelco)filtration column. The gel bed (0.5×4.6 cm) was washed with 20 volumesof 0.01M potassium phosphate pH 7.2. The ascites fluid (0.5 ml),containing ˜5 mg of anti T-2 monoclonal antibody, was applied to thecolumn at a flow rate of 0.6 ml/min. Unbound protein was removed bywashing with the same buffer until baseline absorbance was achieved at280 nm. The T-2 antibody was eluted with 1M NH₄ OH pH 11.6. The pH ofthe fraction was immediately adjusted to 6 with 50% acetic acid, andthen dialyzed for 48 hr vs 2×4 l of 0.01M potassium phosphate, 0.05%NaN₃ pH 7.2. After the elution, the column was washed again with 0.01Mpotassium phosphate pH 7.2 and stored at 4° C. The T-2 antibodyconcentration was determined by measuring the absorbance at 280 nm andcalculating with an extinction coefficient of 1.5 cm² /mg. Molecularweight characterization was performed according to the procedurepreviously described. The integrity of the anti T-2 antibody wasdetermined by the [³ H]T-2 toxin binding assay previously described.

Determination of the titer of T-2 antibody

The affinity purified ascities fluid (0.306 mg/ml) was diluted with0.01M sodium phosphate, 0.9% NaCl, pH 7.2 (1:2, 1:5, 1:10, 1:20, 1:40,1:80 and 1:160). Dextran coated charcoal was prepared by mixing 5 g ofactivated Norit-A charcoal and 0.5 g of Dextran (T-70) in 480 ml of0.01M sodium phosphate, 0.9% NaCl, 0.05% NaN₃, 0.1% BSA pH 7.4.

An aliquot (0.1 ml) from each dilution of antibody was mixed with 0.1 ml(15,000 cpm) of [³ H]T-2 in 0.01M sodium phosphate 0.9% NaCl, 0.05%NaN₃, 0.1% BSA pH 7.4. The tubes were incubated for 2 hr at 37° C., thencooled to 0° C. One ml of ice cold charcoal solution was added and thetubes were vortexed for 30 sec, then placed on ice for 3 min. Aftercentrifugation at 1700×g for 1.5 min at 0° C., 0.5 ml of the supernatantwas pipetted into counting vials containing 3.5 ml of scintillationfluid (Scinti Verse II; Fisher). The samples were counted for 5 min eachin a Packard Tricarb 4530 scintillation counter. The antibody titer wasdetermined by plotting the antibody dilution versus the percent ofradioactivity bound by that dilution. The titer of the T-2 antibody wasfound to be 1:100.

Determination of dissociation rate constant of T-2 antibody

Affinity purified T-2 antibody (1.2 ml; 0.26 mg) was mixed with 1.2 ml(180,000 cpm) [³ H]T-2 in 0.01M NaH₂ PO₄, 0.9% NaCl, 0.1% BSA, 0.05%NaN₃ pH 7.4 and incubated overnight at 4° C. After cooling to 0° C., a0.2 ml sample (0 time) of the above mixture was mixed with 0.2 mldextran coated charcoal (prepared as previously described), andincubated at 0° C. for 10 min. Then the charcoal was removed from thesolution by pushing the fluid through 0.45 μm HA filters (Millipore)with a syringe. A 10,000 fold excess of nonradioactive T-2 was added tothe antibody and [³ H]T-2 mixture. This solution was vortexed, 0.2 mlsamples were taken at 20 sec, 90 sec, 2 min, 4 min, 6 min and 15 min,and the samples were treated in the same manner as the 0 time sample.Seven hundred microliters of each sample solution were pipetted into 10ml of scintillation cocktail. All samples were then counted for 5 min.The dissociation rate constant for T-2 antibody was determined byplotting time vs. log of percent binding using the 0 time sample as 100%binding, and found to be 4.62×10⁻³ sec⁻¹, which corresponds to ahalf-life of 150 seconds.

Analysis of T-2 using an equilibrium assay with affinity separation

Ten microliters of T-2 toxin standards at concentrations of 0, 0.1, and1.0 μg (0, 0.21, and 2.1 nmol) were mixed with 0.2 ml (2.38 nmol) ofFab'-RNase conjugate. This mixture was incubated for 30 min at roomtemperature after which it was applied to a column containing 0.1 ml ofT-2 affinity gel. The column bed (0.5×5 cm) was immediately washed with0.1M TRIS HCl pH 7.5 and eluted with a solution of T-2 (1 mg/ml) in thesame buffer at a flow rate of 0.25 ml/min. Fractions (1.2 ml) werecollected and the RNase activity in each was determined with thepolymeric substrate assay previously described.

Analysis of T-2 by Affinity Chromatography with Fab'-fluorescein

With the necessary reagents prepared and characterized, an analysis forT-2 by the "hit and run" affinity chromatography technique (FIG. 2) wasperformed. A T-2 "hit-and-run" column was packed and conjugate wasloaded as described below. Samples of T-2 (0-50 ng) were applied to thecolumn. After incubation, the amount of fluorescence, due to the releaseof Fab'-fluorescein was measured (FIG. 6). Using this method, 1 ng ofT-2 could be detected. A representative standard curve is shown in (FIG.7). Importantly, all of the data shown in the latter figure, arisingfrom 20 assays, were done successively on the same Fab'-fluorescein T-2hit-and-run column, including a repetition of the 50 ng dose of T-2 atthe end of the assay sequence.

A 1 ml Supelclean (Supelco) filtration column was packed with 0.20 ml ofT-2 affinity gel. The column was equilibrated with 10 ml of 0.01M TRISHCl pH 7.5. One ml (A₂₈₀ =0.8/cm) of Fab'-fluorescein conjugate wasloaded onto the column at a flow rate of 0.1 ml/min. This was followedby washing with equilibration buffer at a flow of 0.3 ml/min whilemonitoring fluorescence by means of a Schoeffel FS970 fluorometer (480nm excitation; 520 nm emission filter). Washing was terminated when thefluorescence signal returned to preloading levels. Samples of T-2 (0, 1,5, 10, 25, and 50 ng) in 0.2 ml of 0.01M TRIS HCl pH 7.5 were applied tothe column at room temperature at a flow rate of 0.68 ml/min. Once thesample completely entered the gel bed the flow was stopped for 5 min. Atthe end of this incubation period, released conjugate was eluted with0.01M TRIS HCl pH 7.5 and the fluorescence signal was measured as justdescribed.

Unsuccessful Analysis of T-2 using an equilibrium assay with affinityseparation

Samples of T-2 were mixed with excess Fab'-RNase, and this mixture wasincubated and then applied to a T-2 affinity column for separation ofbound from free conjugate. Conjugate that is complexed with soluble T-2will not bind to the immobilized T-2 on the affinity gel and thereforewill elute in the wash fractions. Thus, the more T-2 that is present ina sample the more enzyme activity that will elute unretained. In ourhands, however, this assay system was not successful. Highconcentrations of RNase activity were present in the unretainedfractions even in the absence of T-2, and as a result we were unable toquantitate T-2 even at the 1.0 μg level.

Analysis of T-2 by affinity chromatography using Fab'-RNase conjugate

A T-2 affinity column was packed and loaded with Fab'-RNase as describedbelow. Samples (0-3000 ng) of T-2 were applied to the column. Fractionswere collected and the total amount of RNase activity eluted wasmeasured by the polymeric substrate analysis previously described. Onehundred nanograms of T-2 could be detected using this method as shown inFIG. 8.

A T-2 affinity column was packed, loaded with 1 ml (A₂₈₀ =0.8) ofFab'-RNase conjugate, and washed in the same manner as described for theFab'-fluorescein conjugate. The absorbance at 280 nm was monitored andwashing was stopped when it returned to baseline levels. Samples of T-2(0, 100, 250, 500, 1000, and 3000 ng), in 0.2 ml of 0.01M TRIS HCl pH7.5, were applied to the column at a flow rate of 0.68 ml/min. The flowwas stopped and, after a 5 min incubation, released conjugate was elutedwith buffer. Fractions were collected and the amount of eluted RNaseactivity was determined.

Preparation of Fab'-fluorescein-biotin

Fab'-fluorescein (2.8×10⁻⁸ mole), in 0.1M K₂ HPO₄ pH 7.5, was mixed with100 μl of DMSO containing Biotin-X-NHS (2.9×10⁻⁷ mole). This mixture wasincubated overnight on a Nutator at room temperature. Unreacted biotinwas removed by gel filtration on a PD-10 column. The product wascharacterized by determining binding to avidin and T-2.

Preparation of avidin agarose

Two milliliters of Affi-Gel 10 were washed with 6 ml of cold water, and1 ml of avidin solution (4 ng/ml) was added. The gel was incubatedovernight on a Nutator at room temperature, after which the avidinsolution was removed and the gel was washed with 10 vols of 0.1M K₂ HPO₄pH 7.5 and 10 vols of 6M guanidine HCl pH 1.5. The presence of avidinwas established by reaction of the gel with HABA dye.

Determination of Fab'-fluorescein-biotin binding to avidin and T-2

Avidin binding: A column (0.5×1 cm) was packed with avidin agarose. Thecolumn was equilibrated with 0.1M K₂ HPO₄ pH 7.5. Two hundredmicroliters (0.8 A₂₈₀ /ml) of Fab'-fluorescein orFab'-fluorescein-biotin were applied to the column. The column waswashed with 10 ml of 0.1M K₂ HPO₄ pH 7.5, and then eluted with 10 ml of6M guanidine HCl pH 1.5. The amount of fluorescence in the elutedfraction was monitored as previously described.

FIG. 9 depicts the amount of Fab'-fluorescein-biotin bound to an avidinagarose column vs. Fab'-fluorescein as a standard. It can be seen thatthe major portion of Fab'-fluorescein conjugate is not bound to theavidin column but elutes with the wash buffer (0.1M K₂ HPO₄, pH 7.5).The major portion of the Fab'-fluorescein-biotin conjugate binds to theavidin column and is eluted with 6M guanidine HCl pH 1.5.

T-2 binding: The ability of Fab'-fluorescein-biotin to bind to T-2 wasdetermined in comparison to Fab'-fluorescein using a T-2 affinitycolumn. A 1 ml Supelclean (Supelco) column was packed with 200 μl of T-2affinity gel. The gel bed (0.5×1 cm) was equilibrated with 10 mM TrisHCl pH 7.5. One hundred microliters (0.8 A₂₈₀ /ml) of Fab'-fluoresceinor Fab'-fluorescein-biotin were applied to the column. After fiveminutes of incubation at room temperature, the column was washed with 10vols of 10 mM Tris HCl pH 7.5. The column was eluted with a 1 mg/ml T-2solution in the same buffer but with 5% methanol. The amount offluorescence in the eluent was monitored as previously described. Thechromatographic patterns for Fab'-fluorescein-biotin andFab'-fluorescein were essentially the same, as seen in FIG. 10,demonstrating that the binding characteristics ofFab'-fluorescein-biotin for T-2 are intact.

"Hit-and-Run" Immunoassay for T-2 using Fab'-fluorescein-biotinconjugate

The analysis for T-2 using Fab'-fluorescein-biotin conjugate wasidentical to that described above with the fluorescein conjugate excepta 100 ng standard was run as well, and the column was loaded with 1 ml(0.8 A₂₈₀ /ml) of Fab'-fluorescein-biotin conjugate.

A T-2 "hit-and-run' column was packed and loaded withFab'-fluorescein-biotin as described above. Samples of T-2 (0-100 ng)were applied to the column, and the amount of Fab'-fluorescein-biotinreleased after incubation was measured by its fluorescence using aflow-through spectrofluorometer. The data obtained for this conjugatewas similar to that shown previously for the Fab'-fluorescein conjugate.

Preparation of T-2 BSA conjugate

T-2 tresyl ester (12 mg; 0.02 mmol) in 0.5 ml DMSO was added to 5 ml ofa solution of BSA (44.6 mg; 0.66 μmol) in 0.1M potassium phosphate pH7.2. DMSO was added to give a final DMSO/buffer ratio of 4:1. Thereaction mixture was stirred for 48 hr at 4° C., dialyzed at 4° C. vs4×4 l of distilled water, and lyophilized.

The reaction of BSA with T-2 tresyl ester (FIG. 11) resulted in theincorporation of 8 T-2 molecules per molecule of protein (averagevalue). The content of T-2 was determined by calculating the amount ofradioactivity bound per mole of BSA and the concentration of BSA wasbased on its absorbance at 280 nm (E=4.24×10⁴ mol cm⁻¹). Increasing theT-2:BSA ratio in the initial reaction mixture gave rise to insoluble orsparingly soluble products.

Stability of T-2 BSA conjugate

The stability of the T-2 BSA conjugate was investigated by dialyzing theconjugate against 0.01M potassium phosphate buffers ranging in pH from4-10. The number of counts of [³ H]T-2 inside and outside the dialysisbag was determined after 5 and 10 days. In addition, the absorbance at280 nm inside the bag was measured, and the radioactivity to proteinratio determined. The T-2/BSA ratios inside the dialysis bag after 5 to10 days at pH 4, 7, 8, or 10 are shown in Table II. The results showlittle or no hydrolysis of the conjugate even at pH 10.

                  TABLE II                                                        ______________________________________                                        Stability of T-2 Toxin BSA Conjugate by Dialysis                              Against Buffers Ranging From pH 4-10 for 5-10 Days                            Days of Dialysis                                                                     5               10                                                     pH     mols of T-2/1 mol BSA                                                                         mols of T-2/1 mol BSA                                  ______________________________________                                        4      8.8             8.2                                                    7      8.8             8.1                                                    8      7.9             7.5                                                    10     7.7             8.2                                                    ______________________________________                                    

Coupling of BSA-T-2 to Affi-Gel 15

Twenty-one ml of Affi-Gel was drained of isopropyl alcohol and washed 3times with DMSO. To the above gel was added BSA-T-2 solution (9.4 mg in16 ml DMSO) and the mixture was stirred at room temperature for 48 hr.The gel was washed 3 times with DMSO and the unreacted ester was blockedwith 20 ml of 1M ethanolamine washing 3 times for 1.5 hr. The reactionwas followed by measuring absorbance at 260 nm until no more ester wasdetected. The gel was washed with buffer (KPB, 1M, pH 7.2) and stored inthis solvent at 4° C. The concentration of the T-2 on the gel was ≧0.016μmole/1 ml of gel which was determined by its radioactivity (specificactivity of BSA-T-2 is 8 mmole T-2/mmol BSA.)

Reaction of T-2 tresyl ester with hexylamine Sepharose gel

Four milliliters of hexylamine Sepharose gel (6-10 μmole of aminogroups/ml of gel) was washed with 0.5M NaCl, distilled water andDMSO/0.1M KPB, pH 7.2 (70/30(v/v)). To the above gel T-2 tresyl estersolution (40 μmol in 3.9 ml of DMSO-buffer(70/30)) was added and thereaction stirred at 4° C. for 18 hr after which the gel was washed withDMSO/buffer (70/30), MeOH/buffer (70/30) and finally washed with buffer.Twenty μl of the gel was counted which yielded 0.32 μmole T-2/1 ml ofgel.

Preparation of T-2 benzoylhydrazine

T-2 tresyl ester (14 mg, 0.03 mmol) in 1.5 ml of DMSO/buffer (0.01Mpotassium phosphate, pH 7.15, 70/30(v/v)) was added to 0.5 ml ofbenzoylhydrazine solution (11.66 mg, 0.09 mmol) in the same solvent. Thereaction mixture was stirred under N₂ at room temperature for 48 hrs andwas followed by TLC (toluene:ethyl acetate:formic acid (6:3:1)). The T-2tresyl spot disappeared and a faster moving spot appeared. The reactionmixture was poured onto water and the product was extracted into ethylacetate. The organic layer was washed with water and dried overanhydrous Na₂ SO₄. Purification was done by preparative TLC (ethylacetate/hexane (50/50)) which yielded 13 mg of T-2 benzoylhydrazine (77%yield). The structure was confirmed by spectral analysis.

Reaction of epoxy silica gel with adipic acid dihydrazide and couplingto T-2 tresyl ester

Adipic acid dihydrazide (30.88 ng, 177 μmol) was dissolved in 2 ml of 1MKPB pH 7.3 and added to 250 μg of epoxy silica gel (71 μmol epoxy/gm ofgel, Beckman) suspended in 0.5 ml of the same buffer. The suspension wasstirred at room temperature for 72 hrs. After this time the gel waswashed with 0.1M KPB pH 7.3 until no more free amine could be detectedin the wash solution as indicated by the absence of a red color upon theaddition of 3% TNBS. A sample of washed gel was also treated for thepresence of amine groups by means of the TNBS spot test in which a smallamount of gel was suspended in saturated sodium borate buffer pH 9.3 and3% TNBS was added. The appearance of a dark red color on the gel showedthe presence of covalently linked adipic acid hydrazide. T-2 tresylester (41 μmol, specific activity 35 cpm/μg) dissolved in 4 ml ofDMSO/0.1M KPB pH 7.3 (70/30) was added to the remaining gel and thesuspension was stirred at room temperature for 48 hrs. The gel was thenwashed with DMSO/buffer (70/30), MeOH/buffer (30/70), and buffer. Afterwashing, the gel was stored in 1M KPB at pH 6.5. A sample of gel (50 μl)was suspended in 4 ml of scintillation cocktail and counted. Theconcentration of T-2 bound to the gel was calculated from the cpm anddetermined to be ≧2.5 μmol/ml of gel.

The above synthesis is one example of the synthesis of a hydrazidesilica useful in the preparation of conjugates of silica and analytes.More generally, silica possessing epoxy functional groups, e.g.,##STR8## where n is a number from 1 to 10 (preferably 3-6), reacts underthe above conditions with carbohydrazide (H₂ NNH--CO--NHNH₂) or withdihydrazides H₂ N--NH--CO--(CH₂)_(m) --CO--NHNH₂ where m is a numberfrom 1 to 10, to yield hydrazide silica having the generalized formulae##STR9## where n is a number from 1 to 10 and m is a number from 1 to10.

Poor performance of T-2 hexylamine Sepharose and T-2 BSA Affi-Gel 15affinity chromatography columns

While antibody was retained on the T-2 hexylamine Sepharose column, itcould not be eluted either by the addition of T-2 to the column or witha low pH mobile phase. A control experiment with hexylamine Sepharose,not modified with T-2, gave the same result. Thus the antibody wasnonspecifically and strongly bound to this packing, precluding the useof a T-2 hexylamine Sepharose column for affinity purification of theantibody. This column was therefore not suitable for use as ahit-and-run column for assay of T-2.

The performance of the T-2 BSA Affi-Gel 15 column for purification ofantibody was also unsatisfactory. Using T-2 BSA Affi-Gel 15, only 14 to29% of the bound antibody could be eluted by the addition of a 20 foldexcess of T-2 to the column. Antibody also could not be eluted with 0.1MTRIS, 4.5M MgCl₂, pH 7.5. Thus, once again significant nonspecificbinding of the antibody seemed to be taking place on this column. Alsothe amount of antibody eluted with excess T-2 was variable. A T-2 BSAAffi-Gel 15 column therefore was neither attractive for purifying T-2antibody, nor for use in a hit-and-run immunoassay for T-2.

Example 1: Fluorescein-divalent antibody/T-2 tresyl polyacrylamide

1. T-2 antibody-fluorescein conjugate is prepared by reaction of T-2antibody with fluorescein isothiocyanate at pH 9.3.

2. Aminoethyl Bio Gel P2 (Bio Rad) is reacted with succinic anhydride,and then with adipic acid dihydrazide in the presence of EDAC (Bio Rad),forming polyacrylamide adipic acid hydrazide.

3. T-2 toxin is reacted with 6 equivalents of2,2,2-trifluoroethanesulfonyl chloride in acetone/pyridine, forming T-2tresyl ester.

4. Adipic acid hydrazide polyacrylamide is reacted with T-2 tresyl esterin DMSO/buffer pH 7.2 (70/30) to form T-2 polyacrylamide.

5. Fluorescein-T-2 antibody is mixed with T-2-polyacrylamide, followedby washing with buffer at pH 7.5 to remove unreacted antibody, formingT-2 antibody-fluorescein-T-2 polyacrylamide complex.

6. Addition of extrinsic T-2 to this complex releases fluorescein T-2antibody and the eluted fluorescence is measured by a flow-throughfluorescence detector.

7. Alternatively, a DAS antibody may be substituted for T-2 antibody instep 1, giving a DAS antibody-fluorescein conjugate; a DAS tresyl esteris prepared in step 3 and converted to DAS-polyacrylamide in step 4; theDAS antibody-T-2 conjugate is mixed with the DAS-polyacrylamide in step5 providing a DAS antibody-fluorescein-DAS-polyacrylamide complex; andin step 6 addition of extrinsic DAS to this complex releasesfluorescein-DAS antibody for determination of DAS.

Example 2: Fab'-β-galactosidase/T-2 agarose

1. T-2 monoclonal antibody is digested with pepsin, the (Fab')₂ fragmentis reduced with mercaptoethylamine, and the sulfhydryl groups of theresulting Fab' fragments are capped with iodoacetamide according to astandard protocol (Freytag, J. W., Clin. Chem. 30 (1984) 1494),producing protected Fab'.

2. The protected Fab' is reacted with GMBS to produce maleimido-Fab'.

3. β-galactosidase is reacted with the maleimido-Fab', formingFab'-β-galactosidase.

4. Affi-Gel 10 agarose gel (Bio Rad) is reacted with diaminooctane(DAO), followed by reaction with T-2 tresyl ester (prepared as describedin example 1), and capping with ethanolamine, forming T-2-agarose.

5. Fab'-β-galactosidase conjugate is mixed with T-2-agarose, followed bywashing with buffer to remove unreacted conjugate, formingFab'-β-galactosidase T-2 agarose complex.

6. Addition of extrinsic T-2 to the Fab'-β-galactosidase-T-2-agarosecomplex release Fab'-β-galactosidase for detection by its enzymaticactivity with o-nitrophenylgalactoside as the substrate.

7. Alternatively, in step 4 T-2 tresyl ester is immobilized onthiopropyl sepharose 6b (pharmacia), which possesses a reactivesulfhydryl group, forming T-2-agarose with a sulfur atom in the leash;in step 5 this is mixed with Fab'-β-galactosidase and T-2 is determinedas in step 6.

8. Alternatively, adipic acid dihydrazide is substituted for DAO in step4.

Example 3: Fab'-fluorescein/digoxin-silica

1. A monoclonal antibody is obtained by immunizing mice with digoxin-BSAconjugate following a standard protocol (Goding, J. W., MonoclonalAntibodies: Principles and Practices, Academic press, N.Y., 1983).

2. Fab'-fluorescein is produced according to the procedure described inexample 1 except starting with an anti-digoxin antibody.

3. Digoxin is treated with sodium periodate, forming oxidized digoxin.

4. The oxidized digoxin is reacted with ethylenediamine in the presenceof sodium cyanoborohydride, forming amino-digoxin.

5. Amino-digoxin is reacted with epoxy activated silica, formingdigoxin-silica; alternatively, amino-digoxin can be reacted with tresylactivated 2,3-dihydroxypropyl silica (Pierce) to form digoxin-silica;alternatively, oxidized digoxin is reacted with hydrazide silica(prepared as described in example 6), forming digoxin-silica.

6. Fab'-fluorescein is mixed with digoxin-silica to form aFab'-fluorescein digoxin-silica complex.

7. Addition of extrinsic digoxin to the complex releasesFab'-fluorescein which is measured in a flow-through fluorescencedetector.

Example 4: Fab'-HRPO/T₄ Immobilon Membrane

1. T₄ (thyroxine hormone) monoclonal antibody and its maleimido-Fab'fragment are produced as described in example 2, except T₄ -BSA is usedas the immunogen.

2. Horseradish peroxidase (HRPO) is lightly modified with SPDP activeester, forming SPDP-HRPO that retains considerable enzymatic activity.

3. SPDP-HRPO is reduced with one equivalent of dithiothreitol, formingsulhydryl-HRPO, that is in turn reacted with maleimido-Fab', formingFab'-HRPO conjugate.

4. Immobilon membrane is reacted with 1,6-hexanediamine, followed byquenching with ethanolamine, forming aminohexyl-Immobilon membrane.

5. Thyroxine is reacted with acetic anhydride, formingN-acetylthyroxine, which in turn is immobilized onto aminohexylImmobilon membrane by means of the carbodiimide method (Rich, P. H. andSingh, J., The Peptides, 1, (1979) 241-261) forming Acetyl-T₄ -Immobilonmembrane.

6. Alternatively, Immobilon membrane is reacted with ε-amino caproicacid, followed by activation to an NHS ester, forming NHS-Immobilonmembrane, and T₄ or acetyl-T₄ is reacted with NHS-Immobilon membrane toform T₄ -Immobilon membrane or acetyl-T₄ Immobilon membrane.

7. Acetyl-T₄ -Immobilon membrane or T₄ -Immobilon membrane is reactedwith Fab'-HRPO conjugate to form Acetyl-T₄ (or T₄)-Immobilonmembrane-Fab'-HRPO complex, with unreacted Fab'-HRPO being removed bywashing with buffer.

8. Addition of extrinsic T₄ in a stream or by diffusion to eithercomplex releases Fab'-HRPO, which is detected by measuring the enzymaticactivity of released HRPO.

Example 5: [¹²⁵ I] Fab'/Insulin-Nylon Tube

1. Insulin monoclonal antibody and protected Fab' fragment are preparedas described in example 2 except insulin is used as the immunogen.

2. Nylon capillary tubing is activated by hydrolysis in 3M HCl, washingwith water, and treatment with glutaraldehyde as described (Sundaran, P.V., Igloi, M. P., Wasserman, R., Hinsch, W. and Knoke, K. J., Clin.Chem., 24 (1978) 234-239).

3. Protected Fab' is reacted with [¹²⁵ I] Bolton Hunter reagent at pH8.0, forming [¹²⁵ I] Fab'.

4. Insulin is reacted with activated nylon tubing, followed by quenchingof unreacted glutaraldehyde with aqueous ethanolamine, formingnylon-immobilized insulin.

5. [¹²⁵ I] Fab' through the nylon tube, followed by washing awayunreacted [¹²⁵ I] Fab' with buffer, forming [¹²⁵ I] Fab' insulin-nylon.

6. Addition of extrinsic insulin to the complex releases [¹²⁵ I] Fab'from the nylon tube, which is measured by a flow-through radioactivitydetector.

Example 6: Fab'-isoluminol/T-2-silica

1. Anti T-2 Fab' is reacted with N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) forming SPDP-Fab'.

2. Aminobutylethyl isoluminol, prepared as described (Schroeder, H. R.and Yeager, F. M., Anal. Chem., 50, (1978) 1114) is reacted with GMBS toform maleimido-isoluminol.

3. SPDP-Fab' is reduced with dithiothreitol, forming sulfhydryl-Fab',followed by reaction with maleimido-isoluminol, forming Fab'-isoluminol.

4. Epoxy-silica (Beckman) is reacted with adipic acid dihydrazide in 1Mpotassium phosphate buffer, pH 7.3, for 3 days at room temperatue on anutator, giving hydrazide-silica.

5. Hydrazide-silica is reacted with T-2 tresyl ester in 70% DMSO/30%0.01M phosphate buffer pH 7.2 for 48 hr at room temperature, formingT-2-silica.

6. Fab'-isoluminol is reacted with T-2-silica followed by washing withbuffer pH 7.5, forming Fab'-isoluminol-T-2-silica complex.

7. Extrinsic T-2 is added to this complex causing the release ofFab'-isoluminol.

8. Aliquots of the released Fab'-isoluminol are detected by theirchemiluminescence as described (Messeri, G., et al, Clin. Chem., 30(1984) 653).

Example 7: Fab'-isoluminol/E₂ -silica

1. Anti-E₂ (estradiol-17β) monoclonal antibody is prepared as describedin example 2 using an E₂ -BSA conjugate prepared as described (Roda, A.and Belelli, G. F., J. Ster. Biochem., 13 (1980) 449).

2. Anti-E₂ Fab'-isoluminol is prepared as described for Anti T-2Fab'-isoluminol in example 6.

3. E₂ hemisuccinate is prepared as described (Messeri, G., Clin. Chem.,30 (1984) 653), activated with a water soluble carbodiimide, and coupledto hydrazide silica (prepared as described in example 6), forming E₂-silica.

4. E₂ -silica is complexed with Anti E₂ Fab'-isoluminol, followed bywashing, forming Anti E₂ Fab'-isoluminol E₂ silica complex.

5. When this complex is treated with E₂, Fab'-isoluminol is eluted thatis detected by its chemiluminescence as described in example 6.

Example 8: Fab'-dye/DAS glass

1. Fab' recognizing DAS (diacetoxycrirpenol toxin) prepared aspreviously described for Fab' anti T-2 (starting with DAS-BSA immunogenprepared as described below) is reacted with triazine dye at pH 8,forming Fab'-dye conjugate.

2. Glass particles are converted to amino-glass usingβ-aminopropyltriethoxysilane as described (Robinson, P. J., Dunhill, P.and Lilly, M.D., Biochem. Biophys. Acta, 242 (1971) 659-661).

3. Amino glass is activated with glutaraldehyde as described (Robinson,P. J. ibid.), giving glutaraldehyde-glass.

4. DAS tresyl (prepared as described for T-2 tresyl) is reacted withBSA, forming DAS-BSA conjugate.

5. DAS-BSA is reacted with glutaraldehyde glass, forming DAS-glass.

6. Alternatively, glutaraldehyde glass is reacted with BSA or adipicacid dihydrizide, followed by reaction with DAS tresyl ester, followedby quenching of unreacted glutaraldehyde sites with ethanolamine,forming DAS glass.

7. Fab'-dye is reacted with DAS-glass, followed by washing with bufferpH 7.5 to remove any unreacted Fab'-dye, forming Fab'-dye DAS glasscomplex.

8. Addition of extrinsic DAS releases some Fab'-dye conjugate, and theamount of dye released is measured by its absorbance.

Example 9: Fab'-fluorescein/DHT-silica

1. DHT (dihydrotestosterone steriod hormone) is converted to itscorresponding bis-(thioethyl) ketal by reaction with triethylthioborate(B[SCH₂ CH₃ ]₃), forming DHT-TEK.

2. DHT-TEK is activated with tresyl chloride and coupled tohydrazide-silica (prepared as described in example 6), followed byremoval of the two thioethyl groups with mercuric chloride/cadmiumcarbonate in aqueous acetone, forming DHT-silica.

3. Succinyl-DHT is prepared as described (Mickelson, K. E., Teller, D.C., and Petra, P. H., Biochem., 17, (1978), 1409-1415), and coupled toBSA using a water-soluble carbodiimide, giving DHT-BSA.

4. A monoclonal antibody is prepared for DHT using DHT-BSA, and ananti-DHT Fab'-fluorescein is prepared as described in the EXPERIMENTALSECTION for anti T-2 Fab'-fluorescein.

5. DHT-silica is complexed with anti-DHT Fab'-fluorescein, followed bywashing, forming anti DHT Fab'-fluorescein DHT-silica complex.

6. When this complex is treated with DHT, Fab'-fluorescein isspecifically eluted that is detected by its fluorescence.

Example 10: Virus/Polyacrylamide

1. Adipic acid hydrazide polyacrylamide is prepared as in example 1, andrected with glutaraldehyde, forming glutaraldehyde-polyacrylamide.

2. A virus is reacted with glutaraldehyde polyacrylamide, followed byquenching with ethanolamine, forming virus-polyacrylamide.

3. A monoclonal Fab'-fluoresceine is prepared against the virus bystandard techniques, and reacted with the virus-polyacrylamide, forminga Fab'-fluorescein-virus-polyacrylamide complex.

4. Addition of extrinsic virus of Fab'-fluorescein-virus-polyacrylamidecomplex releases Fab'-fluorescein and the eluted fluorescein isdetected.

5. Alternatively, the Fab' is labeled with a release tag (Giese, R. W.,Trends in Anal. Chem. 2 (1983) 166-168, forming Fab'-releasetag-virus-polyacrylamide complex that elutes Fab'-release tag whenexposed to extrinsic virus in a stream, by diffusion or byelectrophoretic migration.

6. Alternatively, the virus is immobilized on Sepharose 2B or Trisacrylby activating these substrates with tresyl chloride following byreaction with the virus, forming virus-Sepharose 2B or virus-Trisacryl,which is used in place of virus-polyacrylamide in the above steps 3-5.

References

1. Bamburg, J. R., Strong, F. M., in "Microbial Toxins," Vol. VII, S.Kadis, A. Ciegler, S. J. Ajl, Eds., pp. 207-292, Academic Press, NewYork, 1971.

2. Bamburg, J. R., in "Mycotoxins and Other Fungal Related FoodProblems," Rodricks, Ed., pp. 144-161, Am. Chem. Soc., Washington, D.C.,1976.

3. Robb, J., and Norval, M., Applied and Environmental Microbiology, 46(1983), 948-950.

4. Ueno, Y. (1980), Adv. Nutr. Res. 3, 301.

5. Bamburg, J. R., Clinical Toxicology, 5 (1972), 495-515.

6. Eppley, R. M., J. Assoc. Off. Anal. Chem., 58 (1975), 906-908.

7. Scott, P. M., J. Assoc. Off. Anal. Chem., 4 (1982), 876-883.

8. Bata, et. al., J. Assoc. Off. Anal. Chem., 66 (1983), 577-581.

9. Chaytor, J. P. and Saxby, M. J., J. Chromatography, 237 (1982),107-113.

10. Chu, F. S., J. Food Protection, 7 (1984), 562-569.

11. Swanson, S. P., et. al., J. Assoc. Off. Anal. Chem., 66 (1983), 909.

12. Fontelo, P. A., Beheler, J., Bunner, D. L., and Chu, F. S., Appliedand Environmental Microbiology, 45 (1983), 640-643.

13. Chu, F. S., Grossman, S., Wel, R. D., and Mirocha, C. J., Appliedand Environmental Microbiology, 37 (1979), 104-108.

14. Hunter, K. W., Brimfield, A. A., Miller, M., Finkelman, F. D., andChu, S. F., Applied and Environmental Microbiology, 49 (1985), 168-172.

15. Freytag, J. W., Dickinson, J. C., and Tseng, S. Y., Clin. Chem., 30(1984), 417-420.

16. Freytag, J. W., Lau, H. P., and Wadsley, J. J., Clin. Chem., 30(1984), 1494-1498.

17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J.Biol. Chem., 193 (1951), 265.

18. Snyder, S. L. and Sobocinski, P. Z. Anal Biochem, 64 (1975),284-288.

19. Sedlak, J. and Lindsay, R. H., Anal Biochem, 25 (1968) 192.

20. Lowry, O. H., et al J. Biol. Chem., 193 (1951) 265-275.

21. Bio Rad Bulletin, 1172, Bio Rad Chemical Division, Richmond, CA.

22. The, T. H. and Feltkamp, T. E. W. Immunol, 18 (1970) 865-873.

23. Smith, P. K., et al Anal Biochem, 150 (1985) 76-85.

What is claimed is:
 1. A hydrazide silica having the formula ##STR10##where X is --NHNHCONHNH₂ or --NHNHCO(CH₂)_(m) CONHNH₂ and n and m arenumbers between 1 and
 10. 2. A hydrazide silica of claim 1 wherein n isa number from 3 to
 6. 3. A hydrazide silica of claim 2 wherein m is 4.